Pone.0008388 1.9

High Prevalence of Multidrug-Tolerant Bacteria andAssociated Antimicrobial Resistance Genes Isolated fromOrnamental Fish and Their Carriage Water
David W. Verner-Jeffreys1*, Timothy J. Welch2, Tamar Schwarz1,3, Michelle J. Pond1, Martin J.
Woodward4, Sarah J. Haig1,3, Georgina S. E. Rimmer1, Edward Roberts1, Victoria Morrison4, Craig
1 Centre for Environment, Fisheries and Aquaculture Sciences, Weymouth Laboratory, Weymouth, Dorset, United Kingdom, 2 United States Department of Agriculture/
Agricultural Research Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, United States of America, 3 Division of Infection and
Immunity, University of Glasgow, Glasgow, United Kingdom, 4 Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom
Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely addedto the water these fish are shipped in to suppress the growth of potential pathogens during transport.
Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp.isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representativeisolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray andconventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriagewater were tolerant to $15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone andfluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR).Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR,blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferasecoding genes were also detected in carriage water samples and bacterial isolates.
Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistantbacteria and resistance genes.
Citation: Verner-Jeffreys DW, Welch TJ, Schwarz T, Pond MJ, Woodward MJ, et al. (2009) High Prevalence of Multidrug-Tolerant Bacteria and AssociatedAntimicrobial Resistance Genes Isolated from Ornamental Fish and Their Carriage Water. PLoS ONE 4(12): e8388. doi:10.1371/journal.pone.0008388
Editor: Stefan Bereswill, Charite´-Universita¨tsmedizin Berlin, Germany
Received October 5, 2009; Accepted October 15, 2009; Published December 21, 2009
Copyright: ß 2009 Crown. This is an open-access article distributed under the terms of the Re-use of Public Sector Information Regulations 2005, which permitunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by The UK government’s Department for Environment Food and Rural Affairs through projects FC1178, FB001 and a sandwichplacement studentship for T.S. (Project FC1172). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
human and animal health [7]. Resistance can either arise frommutations in genes native to the chromosome of the bacterial
The trade in ornamental (pet) fish is greater then 1 billion
species in which they are found, or by acquisition of transferable
animals per year globally [1]. More than 45 million fish per year
genetic elements (e.g. plasmids and/or resistance gene encoding
are imported into the United Kingdom (UK) alone from a wide
integrons) [8]. It is known that either process can lead to the clonal
range of countries, in particular those in South East Asia. An
expansion of resistant pathogens that affect humans and farmed
estimated 14% of all UK households have an aquarium or,
Antibiotic resistance research has typically been very disease-
Antimicrobials are used by owners and retailers to directly
focused, likely contributing to our limited understanding regarding
control bacterial infections [3]. They are also routinely added to
the ecology, evolution and dissemination of antibiotic resistance in
the water these fish are transported in to suppress the growth of
the environment [9]. However, studies have established that
potential pathogens during transport [4]. Trust and Whitby [5]
plasmids, integrons [10] and associated antimicrobial resistance
noted that this practice was widespread more than thirty years ago.
(AMR) genes of bacteria recovered from the aquatic environment
As a consequence, antibiotic tolerant bacteria have likely been
can share very high sequence homology to clinically important
selected for and proliferate in the trade [3,5].
Currently, microbial resistance to antibiotics spans all known
This suggests that there is resistance gene flow between aquatic
classes of natural and synthetic drug agents [6], and bacterial
and anthropogenic sources. The direction of this route of transfer is
resistance to antibiotics continues to pose a serious threat to
unknown, as are the potential health risks arising from this transfer.
December 2009 | Volume 4 | Issue 12 | e8388
Despite the increasing body of evidence regarding the role of
resistance-determining regions of these two genes. Apart from
transferable genetic elements in the dissemination of antibiotic
ampicillin, to which A. hydrophila is intrinsically resistant [23], no
resistance in pathogens that affect farmed fish, there is a relative
antibiotics were included in any of the isolation media used.
paucity of data concerning their role in the development andtransfer of resistance in pet fish. A recent Australian study also
noted a possible a link between the ownership of ornamental fish
Antimicrobial susceptibility was determined for 94 Aeromonas
and a limited number of multidrug resistant (MDR) Salmonella Java
isolates from warmwater species against 34 antibiotics. Methods
for disk-diffusion and broth-microdilution assays followed guide-
It has been recommended that the risks associated with the
lines from the Clinical and Laboratory Standards Institute [24,25].
transfer of antibiotic resistant bacteria through direct contact
SensititreTM panels (Trek Diagnostic systems, UK) were used for
exposure to ornamental fish should be determined [19]. It is also
broth microdilution tests, and antibiotic discs (Abtek Biologicals
important for the ornamental fish industry to recognize the extent
Ltd, Liverpool, UK) for disc-diffusion tests. The MIC values for a
to which the bacteria associated with ornamental fish have
subset of isolates to six antimicrobials (Table S4) were also
determined using laboratory prepared broth-microdilution assays,
Towards these overall aims, a characterization of antibiotic
as recommended by CLSI [25]. The antimicrobials used in testing
tolerance in Aeromonas spp. present in ornamental fish and carriage
were representative of those commonly used to control diseases
water samples was undertaken. Aeromonas spp. were selected as
caused by Gram negative bacteria in human and veterinary
some species, e.g. A. hydrophila, include pathotypes of clinical
medicine, including those used in aquaculture. A total of 33
significance for both fish and humans [20] and are also ubiquitous,
isolates recovered by Cefas between 1992–2004 from coldwater
representative members of the aquatic microbial community. As
species, goldfish (Carassius auratus; 12/33 tested isolates), koi carp
well as determining their tolerance to a range of antimicrobials,
(Cyprinus carpio; 15/33 tested isolates) and other species (6/33),
the presence of select resistance genes in isolates was determined
were also included in the analysis. These species are typically
using a miniaturized microarray. To give an indication of the
reared at temperatures less then 20uC. All historical Aeromonas
extent to which the whole microbial communities present in
isolates were recovered aseptically from the Cefas Bacterial
carriage water may be enriched for genes conferring resistance to
Culture Collection (BCC) held at 280uC in ProtectTM vials,
antimicrobials, a culture-independent characterization of class 1
freeze dried cultures or in liquid nitrogen.
integron diversity in water samples was also undertaken.
In the absence of published resistance breakpoints for Aero-
monads, tolerance to the antimicrobials tested was determined by
examination of the frequency distribution of minimum inhibitoryconcentration (MIC) and disc diffusion diameter values for all the
127 isolates examined. The boundaries of the populations of isolates
A total of 25 consignments, each containing different varieties
showing clearly increased tolerance (non wild type phenotype) and
and species of warm water ornamental fish, were sampled between
those with higher susceptibility were then defined, with those in
February and April 2008. Fish species and associated carriage
between determined as of intermediate (I) susceptibility. Two
waters sampled included, guppies (Poecilia reticulata Peters),
control strains, E. coli ATCC 25922, recommended by both CLSI
threadfin rainbow (Iriatherina werneri Meinken), celebes rainbow
guidelines, and A. hydrophila NCIMB 9240T, were also included in
(Telmatherina ladigesi Ahl), neon gold barb (Puntius semifasciolatus
parallel in all testing. The range of concentrations of antibiotics and
Gu¨nther), harlequin rasbora (Trigonostigma heteromorpha Duncker),
the epidemiological cut-off tolerance values used for both MIC and
neon tetra species (Paracheirodon innesi Myers and Hyphessobrycon
disc diffusion testing are shown in Tables S1 and S2. All tests were
herbertaxelrodi Ge´ry), red wag platy (Xiphophorus maculates), kuhli
performed at 2262uC and results read after between 44–48 h
loach (Pangio kuhlii Valenciennes) and silver molly (Poecilia shenops).
These species are typically reared at temperatures between 24
and 30 uC. The fish were predominantly shipped to the UK fromSingapore (19/25 samples), although wild caught species shipped
Detection of Antibiotic Resistance Genes Using the
from Columbia, Guyana and Brazil were also included. Bags were
Identibac AMR-veTM Miniaturised Micro-Array
either intercepted at the UK’s London Heathrow Airport en route
A total of 23 isolates were analysed for the presence of 54 different
to distributors, or the morning after they arrived in the UK from a
antimicrobial resistance genes using the Identibac AMR-veTM
local wholesale distributor (Weymouth, Dorset). Fish were kept
miniaturised micro-array (http://www.identibac.com/identibac_
and transported to the laboratory in their original carriage water
amr.php). Isolates were analysed following manufacturers instruc-
in sealed bags in boxes, with enough oxygen to survive 24–48 h
tions as previously described [26], with minor modifications. Isolates
before sampling. Samples (10 ml) of carriage water and whole fish
were grown overnight at 22uC on Tryptone Soya Agar. Lysates
homogenised in phosphate buffered saline (PBS) were seeded onto
were prepared by suspending a loopful of culture in 400 ml lysis
solid Aeromonas media (Oxoid, Basingstoke UK). Resultant
buffer (0.1M Tris HCl, 0.005% Tween 20, Proteinase K). This was
presumptive colonies were subcultured and confirmed as Aeromonas
incubated at 65uC for 2 h with regular vortexing, followed by
spp., based on phenotypic testing criteria (Gram negative,
heating to 95uC for 15 min. Approximately two micrograms of
cytochrome oxidase and catalase positive, motile, rods able to
resultant genomic DNA released from the cells were linearly
ferment and oxidize glucose, with API 20NE system (Biomerieux,
amplified using the set of antisense primers provided and
France) biochemical test profiles typical of Aeromonas spp.). A subset
simultaneously biotin labeled. Single-stranded labeled amplified
of 41 isolates, including all those described in Table 1, were further
products were hybridised to the arrays and a signal intensity value
confirmed as Aeromonas spp., based on partial 16S rRNA gene
was determined for each spot on the array by calculating the
sequencing [21]. The gyrA and gyrB genes of a further four of these
quantitative staining value using IconoClust software (version 2;
isolates were partially sequenced, using previously described
CLONDIAG). The mean signal value for the three replicate spots
methods [22]. This was to confirm 16S rRNA gene based
per probe was used for analysis with a signal intensity greater than
identifications and to identify potential mutations in the quinolone
0.3 considered positive, and a signal intensity lower than 0.1 as
December 2009 | Volume 4 | Issue 12 | e8388
December 2009 | Volume 4 | Issue 12 | e8388
negative. Those with an intensity value between 0.1 and 0.3 were
M13F and M13R primers. Clones that contained inserts were
cryopreserved in 50% glycerol. Colonies were then lifted with asterile wooden pick, and stabbed into ampicillin-supplemented
PCR Detection of Antibiotic Resistance Genes, Class 1
lauria agar wells on a 96 well plate. The plate was then incubated
overnight at 37uC prior to transfer to GATC Biotech (Germany)for plasmid extraction and sequencing using M13F and M13R
Water bacterial community DNA samples and isolates were also
directly screened for a range of resistance genes, class 1 integronsand incompatability group (Inc) A/C and IncN plasmids, usingpublished primers and PCR protocols (Table S3). Template DNA
for use in PCR procedures was prepared from isolates by heating
The DNA sequences of a number of the class 1 integrons, tet(A),
colonies suspended in 100 ml molecular grade water at 94uC for
tet(D), tet(E) genes and partial 16S rRNA genes obtained in this
5 min and/or DNAzolTM (Invitrogen) based DNA extraction,
study were deposited in EMBL under the following accession
following the manufacturer’s instructions. The presence of floR,
IncN and IncA/C markers were initially assessed by multiplexPCR utilizing the HotStarTaq Plus Master Mix Kit (Qiagen) and
the primer sets listed in Table S3. PCR conditions were as follows:
5 min activation step at 95uC followed by 35 cycles of 94uC for
Half (47/94) of the isolates recovered from warmwater species
1 min, 55uC for 1 min, 72uC for 1 min, and then a final 10 min
in 2008 were individually tolerant to $15 different antibiotics
(Table 2). This multi-drug tolerance (MDT) was broad ranging,with 64% of the isolates shown to be individually tolerant to
General PCR-Conditions and DNA Sequencing
antimicrobials from seven or more different structural classes of
PCR reaction mixtures (50 ml) generally contained sterile
antimicrobial (Figure 1). Many of the isolates recovered from
molecular-grade water, 1x reaction buffer, 1.5 mM magnesium
coldwater species, were also shown to be MDT, with 27%
chloride, 1.25 U (0.25 ml) Go Taq polymerase (Promega, UK),
individually tolerant to antimicrobials from $3 structural classes
(Figure 1). There were some antimicrobials that most bacteria
50 pmol of each primer. 2.5 ml of template was then added to
tested were highly susceptible to; these included third and fourth
the reaction mixture and samples heated at 94u C for 5 min in a
generation cephalosporins (ceftriaxone, ceftazidime, cefpodoxime,
PTC-225 Peltier thermocycler (MJ Research Inc., Massachusetts,
cefepime and moxalactam) and the carbapenems, imipenem and
USA.). Cycling consisted of 35 cycles of 94uC for 1 min, annealing
meropenem (Table 2). However, some isolates were tolerant to
temperature as indicated for 1 min, 72uC for 1 min, and then a
these antimicrobials, including an A. punctata-like isolate recovered
final 10 min extension at 72uC. All isolates positive for the
from a Singapore guppy sample (Table 1; isolate 08063). This
presence of bla_TEM, florR, qnrS, tet(A), tet(E), tet(D) genes and IncA/
organism was tolerant to 28 of the antimicrobials tested, including
C plasmids were reconfirmed by repeat amplication, in parallel
moxalactam, piperacillin, cefpodixime and imipenem (Table 1).
with previously negative isolates. For isolates positive for floR and
The organism was also determined to have heightened tolerance
IncA/C plasmid markers in the triplex PCR, this was confirmed
to aztreonam (MIC 16 mg l21) and cefepime (MIC 8 mg l21). The
using separate single target PCR. The identity of a number of the
MIC values for six of the antimicrobials were also determined for
resistance genes was also determined by sequencing the resultant
27 isolates, including all those listed in Table 1 (Table S4). Isolates
PCR amplicons. In some cases (for class 1 integrons obtained from
were shown to grow in concentrations of up to 384 mg L21
the isolates and amplicons generated using qnrS primers; Table
ciprofloxacin (3/27 isolates) and oxytetracycline (14/27 isolates). A
S3), PCR products were cloned using the Promega pGEM-T
number of isolates grew in 768 mg L21 of nalidixic acid (15/27
system (Promega, UK). Sequencing was performed either at the
isolates) and oxolinic acid (7/27 isolates). Isolate 08063 also grew
Cefas Weymouth Laboratory, using an ABI 3700 DNA analyser,
in the highest concentrations tested (768 mgL21) for chloram-
or by GATC Biotech Germany (see below).
phenicol and streptomycin (1024 mg L21). A total of 16/27
Sequence data was assembled and initially analysed using the
isolates grew in the highest concentration of suplhadiazine/
Sequencher program (Gene Codes Corp., Ann Arbor, MI, USA).
trimethoprim tested (.730/38.4 mgL21). In total 11/27 of theisolates had MIC values for chloramphenicol of at least 96 mgL21.
Culture-Independent Cloning of Partial Class 1 Integronsfrom Carriage Water Microbial Communities
Miniaturised Microarray and PCR Detection of Antibiotic
For this, approximately 300 ml of each carriage water sample was
Resistance Genes, Class 1 Integrons and Plasmid Markers
vacuum filtered through 0.45 mm (Difco) membranes until saturated
(3–5 filters per sample). Filters were then placed in 25 ml of
A total of 23 isolates were analysed for the presence of 54
molecular grade water (VWR, Leics, UK) in 50 ml falcon tubes
different antimicrobial resistance genes using the Identibac AMR-
(Alpha laboratories, UK). These were then vortexed to resuspend the
veTM miniaturised micro-array. DNA probes for a range of
bacteria. The filters were removed with sterile forceps and the
different resistance genes hybridized with DNA prepared from the
suspension was centrifuged at 3000 g for 15 min. The supernatant
Aeromonas isolates (Table 1). Positive probes included those directed
was discarded and the pellet was resuspended in 200 ml of molecular
at genes mediating resistance to tetracyclines, with samples positive
grade water by vortexing thoroughly. Template DNA was then
by this method for the presence of tet(A), tet(C), tet(D), tet(E) and
extracted using DNAzolTM (Invitrogen) following the manufacturer’s
tet(G). The isolates were also tested in parallel using conventional
instructions. Extractions were stored at 220uC. Partial copies of class
PCR-based detection for tet A-F (Table S3). The presence of tet(A),
1 integrons were PCR-amplified from the carriage water metage-
tet(D) and tet(E) was also confirmed by both PCR and DNA
nomic DNA samples using the primers 5CS/3CS (Table S3).
sequencing in a number of isolates. However there were some
PCR amplicons were cloned as described above. Resultant
discrepancies, with a number of isolates positive for the presence of
clones were screened for the presence of inserts by PCR using
tet genes by miniaturised microarray, even though these genes
December 2009 | Volume 4 | Issue 12 | e8388
Table 2. Proportions (%) of warm water and historical cold water species isolates showing atolerance to 34 differentantimicrobials.
% tolerant isolates (% intermediate tolerant)
% tolerant isolates (% intermediate tolerant)
94 warm water species isolates and 33 coldwater species isolates were tested in total.aTesting was done in compliance with CLSI guidelines (CLSI 2004a; CLSI 2004b). Range of concentrations of antimicrobials tested and interpretative tolerance criteria
bSXT = sulphamethoxazole/trimethoprim.doi:10.1371/journal.pone.0008388.t002
Figure 1. Proportions (%) of isolates recovered from warm water and coldwater species showing tolerance to numbers of differentstructural classes of antimicrobial. Resistance was seen to representatives of the following eleven structural classes: aminoglycocides, second,third and fourth generation cephalosporins, carbapenems, foliate pathway inhibitors, nitrofurans, phenicols, quinolones, fluoroquinolones andtetracyclines. Note, all isolates also displayed expected wild type resistance to penicillins/first generation cephalosporins (not included in figure).doi:10.1371/journal.pone.0008388.g001
December 2009 | Volume 4 | Issue 12 | e8388
could not be detected by PCR (Table 1). These isolates were also
different gene cassettes, including; dihydrofolate reductase types
nearly all tolerant to tetracycline and oxytetracycline (Tables 1
dfrA1, dfrA17, dfrA5, dfrA21, dfrA22, dfrA23; the aminoglycoside
adenyltransferase types aadA1, aadA2; and the quaternary
Genes mediating resistance to betalactams (bla_OXA7 and
bla_TEM1) were detected in many of the isolates by microarray
(Table 3). The 19 other inserts sequenced contained other cloned
and, in the case of bla_TEM1, PCR. A number of isolates were also
sections of microbial community DNA (partial copies of bacterial
positive for the presence of qnrS using the miniaturized
DNA encoding polymerase genes and other bacterial DNA; data
miocroarray. All these isolates and a number of other isolates
were also tested in parallel by PCR for qnrS by PCR, with 47 of 94recently isolated bacteria from warmwater species, shown to be
positive by this alternative method. One organism originallyisolated in 1998 (isolate 98013; Table 1) was also positive. Out of
Other studies have also reported high levels of resistance in
these 48 amplicons, 24 were double-digested with the restriction
bacteria isolated from warmwater ornamental species [3,5].
enzymes HhaI and RsaI and shown to share the same restriction
Tolerance to many of these antimicrobials has likely been driven
profile. Four amplicons were sequenced and shown to share 100%
by their use in the pet fish trade. In particular, oxytetracycline,
identity with a qnrS2 sequence (EU439941), derived from an
nitrofurans (e.g. furazolidone), potentiated suphonamides, and
Aeromonas sp. isolated from the river Seine in France [27].
oxolinic acid, which many of the tested organisms showed high
The florfenicol and chloramphenicol resistance gene floR [28]
tolerance to (Table 2 and Table S4), have been used for many
was detected by miniaturized microarray in three out of 23
years [3]; Cefas Fish Health Inspectorate Staff, Personal
isolates. Follow up PCR analysis confirmed the presence of this
Observations). Two small-scale surveys in the USA in the early
gene in 16 out of 93 recent bacterial isolates from ornamental fish
1990’s also showed a difference in relative tolerance between
species. It was not detected in any of the historical coldwater
isolates recovered from warmwater and coldwater species [3,30].
isolates. Additionally, the floR amplicon was detected in 18/21
Tolerance to tetracyclines was particularly widespread across all
of the carriage water microbial community DNA samples (not
screened isolates (Table 2). It is well established that transferable tet
genes are widely disseminated in the aquatic fish farming
Correlating with observed resistance to aminoglycocides,
environment [11–14,31], and similar genes were identified in
aminoglycoside transferase genes (aadA1, aadA2, aac61b) were
some of the isolates in this study by both PCR and miniaturized
detected in isolates.(e.g. isolates 93024, 08041, 08049, 08063,
microarray. There were also a number of isolates containing DNA
08094 and 08095; Table 1) Miniaturized microarray analysis also
that hybridized with tet probes in the microarray, but were
identified the likely presence of dihydrofolate reductase (dfrA1,
otherwise negative for these and similar genes by PCR. One
dfrA12, dfrA13), as well as sul1, that mediates resistance to
explanation could be that these isolates contained novel variants of
sulmethoprim, in a range of isolates that were resistant to
known tet genes that hybrized with the probes used for
sulphamethoxazole/trimethoprim (Table 1). Fifty percent (56/
miniaturized microarray analysis, but which were not comple-
112) of the isolates tested were also confirmed as positive for class 1
mentary to the PCR primer sets used. More detailed genetic
integrases by PCR. Additionally, DNA sequencing of class 1
characterization, that was beyond the scope of this study (e.g.
integron PCR amplicons from five example isolates identified
whole genome sequencing or tetracycline-directed cloning), is
antibiotic resistance gene cassettes (Table 1). These included
likely required to accurately determine the genetic basis to
confirming the presence of genes also detected by microarray in
tetracycline resistance in these isolates. It should be borne in
these isolates (dfrA1 and dfrA12, aadA1 and aadA2), a gene encoding
mind that the design of the probes on the array was biased toward
a quaterinary ammonium drug pump qacE2, arr2 that mediates
known sequences of Gram negative organisms of human health
rafampacin resistance, as well as the expected int1 gene(Table 1).
Two gene cassettes that encode proteins of unknown function, that
There were also high levels of tolerance observed to all the
have also been identified by other workers in class 1 integrons from
quinolones and fluoroquinolones, particularly in the organisms
a variety of clinical bacterial isolates, were also identified, orfF and
isolated from warmwater species (Table 2; Table S4). It is
orfC. Three of the isolates were also shown to be positive for the
interesting to note that Dixon et al. [3] only reported relatively low
IncA/C plasmid marker by PCR, but none of the isolates were
tolerance to the fluoroquinolone, sarafloxacin, included in that
positive for IncN plasmid markers (Fig. S1). Plasmid markers were
study. Although care should be taken in directly comparing results
detected by PCR in 8/21 (IncN) and 11/21 (IncA/C) carriage
from limited surveys generated using different methodologies, it is
water microbial community DNA samples (not shown).
possible that the tolerance of pet fish associated bacteria to thefluoroquinolones has increased since they were first introduced for
Culture Independent Cloning of Partial Class 1 Integrons
widespread use in clinical and veterinary medicine in the 1980s.
from Carriage Water Microbial Communities
Such tolerance is often mediated by mutations to chromosomal
The class 1 integrons and associated gene cassettes present in
genes [32], with resistance in a number of aquatic bacterial species
the microbial communities in selected water samples were also
linked to changes in the genes coding for DNA gyrase and
examined using a culture-independent approach. Copies of partial
topoisomerase IV enzymes [22,33–34]. The quinolone resistance
class 1 integrons were directly PCR-amplified from water samples,
determining regions of the gyrA and gyrB genes of five
cloned into E. coli and sequenced. The inserts from a total of 58
representative isolates were sequenced (isolates 08020, 08030,
clones obtained from five carriage water samples were sequenced
08033, 08043 and 08094; Table 1), with no coding mutations
and shown to be between 103 and 808 bases in length. Initial
noted, suggesting other mechanisms may be responsible. Trans-
BLAST comparisons [29] with GenBank deposited DNA
ferable, plasmid-mediated resistance is increasingly recognized
sequences determined that 39 of these inserts contained copies
[35]. The finding of qnrS2 [27] at such high prevalence, in
of sections of class 1 integrons. Further comparison with class 1
historical and more recent Aeromonas isolates recovered from
integrons in the Integrall database (http://integrall.bio.ua.pt/)
ornamental fish suggests it may be ubiquitous in bacteria in the
showed that these partial class 1 integrons contained a number of
ornamental fish trade. Its role in observed tolerance to quinolones
December 2009 | Volume 4 | Issue 12 | e8388
Table 3. Resistance gene cassettes identified in copies of class 1 integrons obtained by PCR directly from samples of carriagewater microbial community DNA and cloned into E. coli.
aadA1 (2) aadA2 (2) dfrA5 dfrA17 dfrA27 qacE2 (4)
qacE2 (3) aadA1 (3) dfrA21 dfrA22 (3) dfrA23
Table 3 footnotes.aResistance gene cassettes were identified by comparison with sequences in the integrall database http://integrall.bio.ua.pt/. The sequences of nine of the 38 partial
class 1 integron DNA sequences obtained were deposited in EMBL under the accession numbers FM957877 to FM957885.
baad, aminoglycoside adenylyltransferase, encoding streptomycin-spectinomycin resistance protein; dfr, dihydrofolate reductase genes mediating trimethoprim
resistance; qacE2, gene encoding a quaternary ammonium resistance compound protein (multidrug pump); intI1, integrase: site specific recombination (attI and attCsite).
and fluoroquinolones in isolates carrying the gene is equivocal as
gene cassettes characterized was associated with antibiotic and
this gene typically mediates only low level resistance to these
biocide resistance. This suggests that the carriage water microbial
communities examined were enriched for class 1 integrons
Chloramphenicol and florfenicol tolerance was also observed
containing antimicrobial resistance gene cassettes.
for many of the organisms associated with warmwater species
The detection of IncA/C plasmids in three of the isolates
(Tables 1 and 2). It was also shown that a relatively high
recovered from warm water species was noteworthy. Recent work
proportion of the isolates were also positive for floR. Dissemination
[15–17] has shown that IncA/C plasmids are responsible for self-
of genes conferring resistance to florfenicol is of concern, as in
transmissible antibiotic resistance in North American aquaculture
many countries, including the UK, it has only relatively recently
pathogens, as well as being increasingly important in veterinary
been licensed for use in animals, including fish, destined for
and human medicine [15,40–42]. Preliminary conjugal transfer
human consumption. Many of the isolates also contained genes
experiments, using tetracycline resistance for selection, showed
coding for Beta-lactamases, with blaTEM21 and blaOXA-7 both
successful transfer of IncA/C plasmid markers associated with one
detected (Table 1). Apart from the A. salmonicida isolate tested
of the three positive isolates (isolate 08020; Table 1) to Yersinia
(08078), the Aeromonas spp that were found to carry these two genes
ruckeri and subsequently to E. coli ATCC25922 (using methodology
are typically considered intrinsically resistant to the first generation
described in [15]). In the context of Aeromonas spp. as reservoirs of
cephalosporins and narrow spectrum penicillins they mediate
this clinically important class of plasmids, it is noteworthy that the
resistance to. It is possible that these genes were acquired in
original IncA/C reference plasmid pRA1 was recovered from a
association with functionally more useful genes, coded by plasmids
fish-pathogenic A. hydrophila isolate in 1971 [42,43]. Work assessing
or other transferable elements. As some tolerance was also noted
the importance of plasmid-mediated resistance in the identified
to third generation cephalosporins (Tables 1 and 2), it is suggested
that this be investigated further to determine if this tolerance was
Isolates were identified here that exhibited tolerance to agents
mediated by transferable elements as transfer of these elements
from a number of different structural classes, synthetic (i.e.
nalidixic acid, sulphamethoxazole/trimethorprim) as well as
A total of 50% of the isolates were positive for class 1 integrons,
naturally derived agents, and to relatively new antimicrobials
similar to the levels reported in a survey of motile Aeromonads
recently introduced in human medicine (i.e. ciprofloxacin) (Table
recovered from freshwater fish farms [12] and the 35% reported
S4). Of these resistant isolates, many demonstrated resistance to
for isolates recovered from a slaughterhouse wastewater treatment
multiple antibiotics in the hundreds of mg per liter range, (Table
plant [36]. The proportions of bacteria that were positive for class
S40. These observations suggest a ‘superbug’ phenomenon,
1 integrons appear much lower in the other aquatic environments
whereby multi-antibiotic resistant isolates also demonstrate higher
that have so far been sampled [37–39].
overall resistance levels. Enne et al. [44] postulate that low fitness
Carriage of dfr, sul and aad genes may have contributed to the
costs are associated with multi-antibiotic resistance in E. coli. The
high levels of resistance noted to both foliate pathway inhibitors
authors noted that, once established, combinatorial resistances
and aminoglycocides seen, particularly in the organisms associated
(particularly facilitated via mobile genetic elements such as
with warmwater species (Table 1). Comparisons with sequences in
plasmids) might be difficult to eliminate through reduction in
the Integrall database of integron sequences (http://integrall.bio.
prescribing alone. These results imply that this process may not be
ua.pt/) showed that very similar arrangements of resistance gene
restricted to established pathogenic or opportunistic bacteria, but a
cassettes to those found in bacterial isolates and water microbial
phenomenon common in environmental bacteria, or bacteria with
communities have previously been described in class 1 integrons
established environmental reservoirs.
found in other human, fish and terrestrial animal pathogens.These included those associated with clinical and environmental
Aeromonas isolates from Taiwan [20].
A surprisingly high level of antimicrobial tolerance was
All 39 of the class 1 integrons identified in the constructed clone
identified in bacteria associated with warmwater ornamental
library contained gene cassettes that have also been recovered
species and ornamental fish carriage water. The significance of
from human and veterinary clinical isolates. All but one of the 40
these tolerant bacteria from ornamental fish in acting as a
December 2009 | Volume 4 | Issue 12 | e8388
potential reservoir for mobilisable antibiotic resistance should be
Interpretative tolerance cut offs and range of
systematically assessed. This should help prevent the potential
concentrations of discs used for disc diffusion testing [25].
spread of resistance to pathogens of human and animal health
Found at: doi:10.1371/journal.pone.0008388.s003 (0.03 MB
importance, and improve fish welfare and treatment. Antibiotic
use for prophylactic purposes should also ideally be replaced by
Primers and PCR conditions used in study.
better husbandry and transport conditions and the use of
Found at: doi:10.1371/journal.pone.0008388.s004 (0.03 MB
MIC values (IˆJg/ml) determined for selected isolates
Multiplex detection of the florfenicol resistance gene,
Found at: doi:10.1371/journal.pone.0008388.s005 (0.06 MB
floR, and markers for the IncA/C and IncN plasmids.
Found at: doi:10.1371/journal.pone.0008388.s001 (0.46 MB PPT)
Tolerance cut off values and range of concentrations
of antimicrobials used in broth microdilution testing [24]. Also
Jennifer Harper provided valuable and skillful technical assistance.
shown are the ranges in MIC values recorded for the two controlstrains included in parallel during testing, E. coli NCIMB 25922
Conceived and designed the experiments: DWVJ TJW TS MJP MW CBA.
Found at: doi:10.1371/journal.pone.0008388.s002 (0.05 MB
Performed the experiments: DWVJ TJW TS MJP GSER SJH ER VM.
Analyzed the data: DWVJ TJW TS MJP MW GSER SJH ER VM CBA.Wrote the paper: DWVJ TJW TS CBA.
1. Wittington RJ, Chong R (2007) Global trade in ornamental fish from an
19. Anon (1999) Report on Microbial Resistance in Relation to Food Safety. Advisory
Australian perspective: The case for revised import risk analysis and
Committee on the Microbiological Safety of Food. London. 1999. HMSO.
management strategies. Prev Vet Med 81: 92–116.
20. Chang Y, Shih DY, Wang J, Yang S (2007) Molecular characterization of class 1
2. OATA website http://www.ornamentalfish.org/aquanautmarket/market.php)
integrons and antimicrobial resistance in Aeromonas strains from foodborne
outbreak-suspect samples and environmental sources in Taiwan. Diagn Micr
3. Dixon BA, Yamashita J, Evelyn F (1990) Antibiotic resistance of Aeromonas spp.
isolated from tropical fish imported from Singapore. J Aquat Anim Health 2:
21. Pond MJ, Stone DM, Alderman DJ (2006) Comparison of conventional and
molecular techniques to investigate the intestinal microflora of rainbow trout
4. Cole B, Tamaru CS, Bailey R (1999) Shipping practices in the Ornamental Fish
Oncorhynchus mykiss). Aquaculture 261: 194–203.
Industry. Center for Tropical and Subtropical Aquaculture, Publication No. 131
22. Gon˜i-Urriza M, Arpin C, Capdepuy M, Dubois V, Caumette P, et al. (2002)
Type II topoisomerase quinolone resistance-determining regions of Aeromonas
5. Trust, TJ, Whitby, JL (1976) Antibiotic resistance of bacteria in water containing
caviae, A. hydrophila, and A. sobria complexes and mutations associated with
ornamental fishes. Antimicrob Agent Chemother 10: 598–603.
quinolone resistance, Antimicrob Agent Chemother 46: 350–359.
6. D’Costa V, McGrann KM, Hughes DW, Wright GD (2006) Sampling the
23. Rossolini GM, Walsh T, Amicosante G (1996) The Aeromonas metallo-beta-
antibiotic resistome. Science 311: 374–377.
lactamases: genetics, enzymology, and contribution to drug resistance. Microb
7. Levy, SB, Marshall B (2004) Antibacterial resistance worldwide: causes,
challenges and responses. Nature Med Rev 10: S122–S129.
24. Clinical and Laboratory Standards Institute (2004a) Methods for Broth dilution
8. O’Brien TF (2002) Emergence, spread and environmental effect of antimicrobial
Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Proposed
resistance: how use of an antimicrobial anywhere can increase resistance to any
Guideline, M49-P. CLSI, Wayne, PA, USA.
antimicrobial anywhere else. Clin Infect Dis 34(Suppl 3): S78–S84.
25. Clinical and Laboratory Standards Institute (2004b) Methods for Antimicrobial
9. Singer R, Ward MP, Maldonado G (2006) Can landscape ecology untangle the
Disk Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Proposed
complexity of antibiotic resistance? Nature Rev. Microbiol 4: 943–952.
Guideline, M42-P. CLSI, Wayne, PA, USA.
10. Hall RM, Collis CM, Kim M J, Partridge SR, Recchia GD, et al. (1999) Mobile
26. Batchelor R, Hopkins K L, Liebana E, Slickers P, Ehricht R, et al. (2008)
gene cassettes and integrons in evolution. Ann N Y Acad Sci 870: 68–80.
Development of a miniaturised micro-array for the rapid identification of
11. Rhodes G, Huys G, Swings J, McGann P, Hiney M, et al. (2000) Distribution of
antimicrobial resistance genes in Gram-negative bacteria. Int J Antimicrob Ag
oxytetracycline resistance plasmids between Aeromonads in hospital and
aquaculture environments: implication of Tn1721 in dissemination of the
27. Cattoir V, Poirel L, Aubert C, Soussy C-J, Nordmann P (2008) Unexpected
tetracycline resistance determinant tetA. Appl Environ Microbiol 66: 3883–3890.
occurrence of plasmid-mediated quinolone resistance determinants in environ-
12. Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL (2001) Incidence, distribution
mental Aeromonas spp. Emerg Infect Dis 14: 2008 Feb Available: http://www.
and spread of tetracycline resistance determinants and integron- associated
cdc.gov/EID/content/14/2/231.htm. Accessed 1 July 2009.
antibiotic resistance genes among motile Aeromonads from a fish-farming
28. Smith P (2008) Aquaculture and florfenicol resistance in Salmonella enterica
environment. Appl Environ Microbiol 67: 5675–5682.
Typhimurium Dt104. Emerg Infect Dis 14, 8: 1327. Available http://www.cdc.
13. Furushita M, Shiba T, Maeda T, Yahata M, Kaneoka A, et al. (2003) Similarity
gov/EID/content/14/8/1327b.htm Accessed October 5, 2009.
of tetracycline resistance genes isolated from fish farm bacteria to those from
29. Altschul SF, MaddenTL, Scha¨ffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
clinical isolates. Appl Environ Microbiol 69: 5336–5342.
BLAST and PSI-BLAST: a new generation of protein database search
14. Sørum H, L’Abe´e-Lund TM, Solberg A, Wold A (2003) Integron-containing
programs. Nucleic Acids Res 25: 3389–3402.
IncU plasmids pRAS1 and pAr-32 from the fish pathogen Aeromonas salmonicida.
30. Dixon BA, Issvoran G (1993) Antibacterial drug resistance in Aeromonas spp.
Antimicrob Agents Chemother 47: 1285–1290.
isolated from domestic goldfish and koi from California. J. World Aqua Society
15. Welch TJ, Fricke WF, McDermott PF, White DG, Rosso M-L, et al. (2007)
Multiple Antimicrobial Resistance in Plague: An Emerging Public Health Risk.
31. Jacobs L, Chenia HY (2007) Characterization of integrons and tetracycline
PLoS ONE 2(3): e309. doi:10.1371/journal.pone.0000309.
resistance determinants in Aeromonas spp. isolated from South African
16. Welch TJ, Evenhuis J, White DG, Patrick F, McDermott PF, et al. (2008) IncA/
aquaculture systems. Int J Food Microb 114: 295–306.
C plasmid-mediated florfenicol resistance in the catfish pathogen Edwardsiella
32. Ruiz J (2003) Mechanisms of resistance to quinolones: target alterations,
ictaluri. Antimicrob Agent Chemother 53: 845–846.
decreased accumulation and DNA gyrase protection. J Antimicrob Chemother
17. McIntosh D, Cunningham M, Ji B, Fekete FA, Parry EM, et al. (2008)
Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian
33. Giraud E, Blanc G, Bouju-Albert A, Weill F-X, Donnay-Moreno C (2004)
isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an
Mechanisms of quinolone resistance and clonal relationship among Aeromonas
IncA/C plasmid similar to the Salmonella enterica plasmid pSN254. J Antimicrob
salmonicida strains isolated from reared fish with furunculosis. J Med Microbiol
18. Musto J, Kirk M, Lightfoot D, Combs, Mwari L (2006) Multi drug resistant
34. Izumi S, Aranishi F (2004) Relationship between gyrA mutations and quinolone
Salmonella Java infections acquired from tropical ornamental fish aquarium,
resistance in Flavobacterium psychrophilum isolates. Appl Environ Microbiol 70:
Australia, 2003-04. Commun Diseas Intell 30: 222–227.
December 2009 | Volume 4 | Issue 12 | e8388
35. Robicsek A, Jacoby GA, Hooper DC (2006) The worldwide emergence of
40. Egorova S, Timinouni M, Demartin M, Granier SA, Whichard JM, et al. (2008)
plasmid-mediated quinolone resistance. Lancet Infect Dis. 6: 629–640.
Ceftriaxone- resistant Salmonella enterica Serotype Newport, France. Emerging
36. Moura A, Henriques I, Ribeiro R, Correia A (2007) Prevalence and
Infectious Diseases 14: 954–957. http://www.cdc.gov/eid/content/14/6/954.
characterization of integrons from bacteria isolated from a slaughterhouse
wastewater treatment plant. J Antimicrob Chemother 60: 1243–1250.
41. Pan JC, Ye R, Wang HQ, Xiang HQ, Zhang W, et al. (2008) Vibrio cholerae O139
37. Rosser SJ, Young HK (1999) Identification and characterisation of class 1
multiple drug resistance mediated by Yersinia pestis pIP1202-like conjugative
integrons in bacteria from an aquatic environment. J Antimicrob Chemother 44:
plasmids. Antimicrob Agents Chemother 52: 3829–3836.
42. Fricke WF, Welch TJ, McDermott PF, Mammel MK, LeClerc JE, et al. (2009)
38. Gaze, WH, Abdouslam N, Hawkey PM, Wellington EMH (2005) Incidence of
Comparative genomics of the IncA/C multidrug resistance plasmid family.
class 1 integrons in a quaternary ammonium compound-polluted environment.
Antimicrob Agents Chemother 49: 1802–1807.
43. Aoki T, Egusa S, Ogata Y, Watanabe T (1971) Detection of resistance factors in
39. Gillings MR, Krishnan S, Worden PJ, Hardwick SA (2008) Recovery of diverse
fish pathogen Aeromonas liquefaciens. J Gen Microbiol 65: 343–349.
44. Enne VI, Livermore DM, Stephens P, Hall LM (2001) Persistence of
genes for class 1 integron-integrases from environmental DNA samples. FEMS
sulphonamide resistance in Escherichia coli in the UK despite national prescribing
restriction. Lancet 357: 1325–1328.
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